For individuals with Type 1 diabetes (T1DM) hypoglycemia remains the principal factor limiting the achievement of near-normal glucose control, which has been shown to reduce the microvascular complications of diabetes. This proposal aims to investigate the role of AMP-activated protein kinase (AMPK) in the ventromedial hypothalamus (VMH) in the mechanisms used by the brain to sense a falling glucose and to trigger glucose counterregulatory responses. It will also determine whether alterations in AMPK expression and activity may underlay the defects in hypoglycemia couterregulation that develop following recurrent hypoglycemia. In vivo hyperinsulinemic hypoglycemic clamps studies following microinjection of an AMPK activator to the VMH will be performed in (i) Sprague-Dawley (SD) rats to determine whether this results in the generation of a neural signal to the liver to stimulate hepatic glucose production, (ii) SD rats who have undergone 3 days of recurrent hypoglycemia to determine whether it is possible to reverse the defects in hormonal counterregulation that result from recurrent hypoglycemia by further stimulating AMPK. Additional, in vivo hyperinsulinemic glucose clamp studies following VMH microinjection of varying amounts of AMPK activator will be performed to investigate the dose-response nature of AMPK activation during hypoglycemia, and the effect of recurrent hypoglycemia on this signaling system. To support the in vivo studies in vitro Westem, and AMPK activity, analyses obtained from micropunches of rat VMH under varying physiological conditions will be obtained. Finally, the proposal aims to obtain data that will support the hypothesis that recurrent hypoglycemia induces mitochondrial biogenesis in the VMH, an effect that may be mediated by AMPK.